Nano-IR technology was firstly employed on bamboo fiber research with purpose to further understand the fine structure of bamboo fiber cell wall. Chemical constituent distribution of bamboo fiber was studied, and the feasibility of the novel technology was discussed by comparing with other traditional methods. The results showed that Nano-IR technology, which has made a breakthrough on diffraction limit of traditional infrared spectroscopy, can acquire nano-scale infrared spectrum of bamboo cell wall in situ condition. The characteristic peak positions of Nano-IR spectrum is basically the same with that of microscopic FTIR spectrum, indicating that Nano-IR spectrum can reveal the chemical information of bamboo cell wall. The results of the present work suggested that nano-IR technology could be an effective research tool in research of nano chemical composition distribution of bamboo cell wall.